赤霉素对精子运动力和细胞凋亡的影响及其机制

EFFECTS OF GIBBERELLIN ON HUMAN SPERM MOTILITY AND APOPTOSIS IN VITRO AND ITS MECHANISM

目的探讨赤霉素对精子运动力和细胞凋亡的影响及其机制。方法采用Percoll梯度离心法提取有正常生理功能的人精子作为正常精子模型,提取的优质精子培养于含不同浓度赤霉素的G-IVF PLUS培养液中,采用精子质量分析仪检测精子运动力;流式细胞分析仪检测精子细胞活性氧(ROS)水平及凋亡情况;免疫荧光技术检测氧化应激、凋亡相关蛋白表达量;ATP酶检测试剂盒检测Na^+-K^+-ATP酶和Ca^(2+)-ATP酶活性。结果精子运动力随赤霉素浓度的增加、时间的延长逐渐降低;处理组精子细胞ROS水平明显高于对照组(F=21.40,P<0.05),过氧化物歧化酶(SOD)蛋白水平较对照组明显降低(F=28.13,P<0.05);凋亡蛋白P53、BAX表达水平较对照组明显升高(F=104.54、59.29,P<0.05);Na^+-K^+-ATP酶和Ca^(2+)-ATP酶活性较对照组明显降低(F=24.48、18.81,P<0.05)。结论赤霉素可通过氧化应激反应降低精子运动力,促进精子细胞凋亡。

Objective To investigate the effects of gibberellin on human sperm motility and apoptosis in vitro and its mechanism. Methods The human sperms with normal physiological function were selected as normal control group using a discontinuous Percoll gradient centrifugation（45%-90%）.The high-quality sperms（treatment group）were exposed to G-IVF PLUS medium with different concentrations of gibberellin in vitro.The sperm motility was measured by a sperm quality analyzer at different time points.Flow cytometry was used to determine the level of reactive oxygen species（ROS）and apoptosis.Immunofluorescence was used to measure the expression of proteins related to oxidative stress and apoptosis.The activity of Na＋-K＋-ATP enzyme and Ca^（2＋）-ATP enzyme were determined by an ATP enzyme assay kit. Results The sperm motility gradually decreased with time and the increase in gibberellin concentration.Compared with the control group,the treatment group had a significantly higher ROS level（F=21.40,P〈0.05）and a significantly lower level of superoxide dismutase（F=28.13,P〈0.05）;moreover,the treatment group had significantly higher expression of P53 and BAX（apoptotic proteins）（F=104.54,59.29;P〈0.05）and significantly lower activity of Na＋-K＋-ATP enzyme and Ca^（2＋）-ATP enzyme（F=24.48,18.81;P〈0.05）. Conclusion Gibberellin can reduce sperm motility and promote sperm apoptosis via oxidative stress reaction.